PROJECT SUMMARY Genomic analysis suggests that up to 50% of High Grade Serous Ovarian Cancers (HGSCs) harbor a genomic alteration that might confer a DNA damage repair defect, making therapies that target such defects potential treatment options. There is no method to predict which patients will respond to such therapies, which is a major problem in the field. Preliminary data indicate that patient derived HGSC organoids may be a faithful model system in which to perform functional assays to predict patient therapeutic response. Data from a limited analysis of HGSC organoids suggest that stalled replication fork protection defects are more common than homologous recombination defects in HGSC and that more patients may benefit from the wider array of therapies available to target such defects, including carboplatin, gemcitabine, ATR, WEE1, and CHK1 inhibitors. The goal of this mentored research career development proposal is to utilize patient derived HGSC organoid cultures to understand the prevalence, mechanisms, and therapeutic relevance of stalled replication fork protection defects in HGSC. The proposed research studies encompass multiple disciplines including molecular biology, DNA sequencing, and animal modeling which will help investigate the role of stalled replication fork protection defects in HGSC and also provide a well-rounded career development strategy for becoming scientifically independent through execution of the following specific aims: Specific Aim 1: Assess the prevalence of fork protection defects in HGSC and whether fork protection defects predicted by HGSC organoid functional assays lead to therapeutic sensitivity to carboplatin and ATR, WEE1, and CHK1 inhibitors. This will be accomplished by generating organoids from patients being treated with carboplatin and ATR, WEE1, and CHK1 inhibitors, performing functional assays to assess stalled fork protection capacity and therapeutic sensitivity of the organoids in parallel to sequencing analysis, and comparing organoid and patient outcomes. Specific Aim 2: Uncover mechanisms leading to fork instability in the organoids and whether different mechanisms of fork protection defects lead to differing sensitivities to the above agents. This will be accomplished using molecular and cellular biology analysis of specific pathways in the organoids. Specific Aim 3: In vivo validation of in vitro mechanisms of stalled replication fork protection defects leading to therapeutic responses in organoid xenograft models of HGSC. This will be accomplished by generating mouse models using select organoids from aim 2 and testing them for therapeutic responses to agents used in aim 2. The career development award candidate is an MD/PhD clinically trained in anatomic pathology. The proposed research will occur at Dana-Farber Cancer Institute under the mentorship of Dr. Alan D'Andrea. The candidate will utilize the additional training provided by this award to facilitate her ultimate career goal of becoming an independent physician scientist and leader in the field of ovarian cancer.